Structure activity relationships of fused bicyclic and urea derivatives of spirocyclic compounds as potent CCR1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2013.11.062

A series of fused bicyclic and urea derivatives of spirocyclic compounds were designed, synthesised and evaluated in vitro as potent CCR1 antagonists. In particular, 4 (7 nM), 44 (1.3 nM), 48 (0.89 nM) and 50 (0.63 nM) were the most potent hCCR1 antagonis

Full text of DOI:10.1016/j.bmcl.2013.11.062

Bioorganic & Medicinal Chemistry Letters 24 (2014) 108–112
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure activity relationships of fused bicyclic and urea derivatives
of spirocyclic compounds as potent CCR1 antagonists
b,
Nafizal Hossain a, , Marguérite Mensonides-Harsema , Martin E. Cooper a, Tomas Eriksson b,
,
⇑
Svetlana Ivanova b, , Lena Bergström a
a Department of Medicinal Chemistry, Respiratory, Inflammation and Autoimmunity Innovative Medicines, AstraZeneca R&D, Pepparedsleden 1, SE-431 83 Mölndal, Sweden
b Department of Medicinal Chemistry, AstraZeneca R&D Lund, Scheelevägen 1, SE-221 87 Lund, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of fused bicyclic and urea derivatives of spirocyclic compounds were designed, synthesised and
evaluated in vitro as potent CCR1 antagonists. In particular, 4 (7 nM), 44 (1.3 nM), 48 (0.89 nM) and 50
(0.63 nM) were the most potent hCCR1 antagonists in this series of compounds. Moreover, some of these
substances demonstrated good rodent cross-over, especially 46 which exhibited very high rat CCR1 bind-
ing affinity with an IC50 value of 16 nM.
Received 1 November 2013
Revised 19 November 2013
Accepted 25 November 2013
Available online 4 December 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Chemokines
Antagonists
CCR1
Spirocyclic
Receptor
Chemokines belong to a family of cytokines which play an
important role in various inflammatory diseases by mediating leu-
kocyte activation and recruitment.1–4 They bind to their target
receptors on the cell surface of leukocytes and transmit intracellu-
lar signals through the activation of guanine nucleotide regulatory
proteins (G-proteins), resulting in movement of the leukocytes
towards the source of the chemokines and the desired site of
action. Chemokine receptors are thus attractive targets in the
development of drugs against autoimmune/inflammatory dis-
eases5–8 due to their vital role in regulation of leukocyte trafficking.
The C–C (cystine–cystine) chemokine receptor-1 (CCR1) and its
exhibited both high binding affinity and cell based functional
potency.10 These compounds contain either a N-acetyl (1a–d) or
a benzamide (1e–f) moiety as part of a substituted phenyl group.
The N-acetyl moiety was important for CCR1 activity but has the
potential to be problematic due to possible formation of toxic ani-
line metabolites. The replacement of the N-acetyl moiety with the
benzamide moiety in a substituted phenyl moiety also resulted in
potent CCR1 antagonists (Fig. 1).10b
However, these compounds are prone to inhibit the hERG chan-
nel10b which could render them unsuitable as potential drugs. It
was found that the hERG channel activity could be eliminated by
the introduction of an acidic group but the permeability was mod-
est for such zwitterionic compounds.10c We therefore postulated
that structural changes which would shift the balance between
permeability and hERG activity might provide a way forward for
this class of compounds. Thus the N-acetyl series 1a–d (Fig. 1)
was revisited to determine wheather the N-acetyl group could be
masked (for instance by incorporation into a ring) or made meta-
bolically more stable, with a view to minimising the risk of aniline
metabolite formation. To this end, a series of fused bicyclic com-
pounds as well as a series of urea derivatives were designed and
these were synthesised according to the procedures outlined
below.
major endogenous ligands MIP-1a (CCL3) and RANTES (CCL5) play
an important role in chronic inflammatory diseases such as multi-
ple sclerosis5,6 and rheumatoid arthritis.7,8 Thus, inhibition of CCR1
is expected to be beneficial for patients who suffer from such
inflammatory disorders. Small molecule CCR1 antagonists could
therefore be useful as therapeutic agents and the search for specific
and highly potent CCR1 antagonists has recently been highlighted
in the literature.9 Herein, we describe the synthesis and biological
evaluation of a series of fused bicyclic and urea derivatives of spi-
rocyclic compounds as potent CCR1 antagonists.
Recently, we reported a series of conformationally constrained
spirocyclic compounds represented by 1a–f (Fig. 1) many of which
Compounds 4–6 were synthesised as described in Scheme 1.
The epoxide opening reaction of (R)-2-(chloromethyl)oxirane with
spirocyclic amine 210a,11 in ethanol at room temperature yielded an
intermediate chlorohydrin which was treated in situ with NaOMe
⇑
Corresponding author. Tel.: +46 317761851; fax: +46 317763818.
E-mail address: nafizal.hossain@astrazeneca.com (N. Hossain).
Former employee.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.11.062

N. Hossain et al. / Bioorg. Med. Chem. Lett. 24 (2014) 108–112
109
O
OH
N
O
R1
R2
R
OH
O
O
N
O
N
O
X
Cl
F
1a: X = Cl, R = OH, R1 = NHAc,R2 = OH, IC50 = 0.36 nM
1b: X = F, R = OH, R1 = NHAc, R2 = OH, IC50 = 0.52 nM
1c: X = F, R = NH2, R1 = NHAc, R2 = OH, IC50 = 1.4 nM
1d: X = Cl, R = OH, R1 = NHAc, R2 = H, IC50 = 0.96 nM
1e: X = Cl, R = OH, R1 = -CONHCH3, R2 = OH, IC50 = 1.8 nM
1f: IC50 = 1.4 nM
Figure 1. Previously reported CCR1 antagonists.10
O
O
O
(a)
(b)
N
N
N
Cl
Cl
Cl
3: (71%)
2
O
O
OH
HN
OH
HN
O
O
N
O
X
O
Cl
4: X = O (5%)
6: (38%)
5: X = CH2 (25%)
Scheme 1. Reagents and conditions: (a) (R)-2-(chloromethyl)oxirane, EtOH, rt, 36 h, then NaOMe/MeOH, rt, 1 h; (b) 5-hydroxy-2H-1,4-benzoxazin-3 (4H)-one or 8-hydroxy-
3,4-di-hydroquinolin-2 (1H)-one or 8-hydroxy-quinolin-2 (H)-one, K2CO3, DMF, 110 °C, 24 h (5–38%).
in methanol at room temperature to afford the intermediate spiro-
cyclic epoxide 3 in good yield. Treatment of epoxide 3 with either
of epoxides 14, 15 and 16, respectively. These epoxides were
opened with spirocyclic amine 2 in ethanol at 78 °C to give target
compounds 17, 18 and 19, respectively in acceptable yields.
To investigate the effect of replacing the carbon linker hydroxyl
group with a primary amino group, as in the previously reported
compound 1c, the fused bicyclic compounds 26 and 28 were de-
signed and synthesised according to the procedure outlined in
Scheme 4. Thus spirocyclic amine 2010a was alkylated with 21
in the presence of Et3N in dichloromethane at 0 °C to afford 22 in
59% isolated yield. The ester derivative 22 was reduced to the cor-
responding alcohol 23 in acceptable yield by treatment with LiBH4
in THF at 0 °C to room temperature, and this was converted to the
nosylate 24 in modest yield, by treatment with 3-nitrobenzenesul-
phonyl chloride in dichloromethane in the presence of Et3N.
Compound 24 was treated with 5-hydroxy-2H-1,4-benzoxazin-3
(4H)-one in the presence of Cs2CO3 in DMF to give 25 which was
subsequently treated with trifluoroacetic acid in dichloromethane
to afford 26 in 21% isolated yield over two steps. Treatment of 23
5-hydroxy-2H-1,4-benzoxazin-3-(4H)-one,
8-hydroxy-3,4-dihy-
droqui-nolin-2-(1H)-one or 8-hydroxy-quinolin-2-(1H)-one in the
presence of K2CO3 in DMF at 110 °C for 24 h gave 4, 5 and 6, respec-
tively in low yields. The urea analouge 9 was synthesised as out-
lined in Scheme 2. The reaction of 2-amino-3-hydroxybenzoic
acid with urea in NMP under microwave irradiation at 200 °C
yielded 8-hydroxyquinazolin-2,4-(1H,3H)-dione 8 which was cou-
pled with epoxide 3 in the presence of of K2CO3 in DMF at 110 °C to
give 9 in very low yield.
In view of the low yields for the above coupling steps an alter-
native procedure, whereby the epoxide intermediates were formed
from the phenol derivatives as described in Scheme 3, was used for
the synthesis of subsequent compounds. Thus, for the synthesis of
5-6-fused compounds, the phenols 10, 11 and 12 were treated sep-
arately with (2S)-oxiran-2-yl-methyl-3-nitrobenzene sulphonate
in the presence of K2CO3 in DMF at 110 °C to afford high yields
O
O
NH
HN
NH2
O
NH
OH
HN
O
(a)
(b)
HO
HO
N
O
O
O
O
Cl
9: (3%)
7
8: (29%)
Scheme 2. Reagents and conditions: (a) Urea, NMP, 200 °C microwave oven, 20 min (29%); (b) 3, K2CO3, DMF, 110 °C, 24 h (3%).

110
N. Hossain et al. / Bioorg. Med. Chem. Lett. 24 (2014) 108–112
z
z
X
X
NO2
O
Y
Y
O
S
HO
O
O
(a)
(b)
O
O
13
14: X = CH, Y = O, Z = CH3 (78%)
15: X = N, Y = S, Z = NH2
16: X = N, Y = S, Z = CH3 (71%)
10: X = CH, Y = O, Z = CH3
11: X = N, Y = S, Z = NH2
12: X = N,, Y = S, Z = CH3
z
OH
X
O
Y
N
O
Cl
17: X = CH, Y = O, Z = CH3 (59%)
18: X = N, Y = S, Z = NH2 (23% over two steps)
19: X = N, Y = S, Z = CH3 (57%)
Scheme 3. Reagents and conditions: (a) K2CO3, DMF, 110 °C, 24 h; (b) spirocyclic amine 2, EtOH, 78 °C 4 h (23–78%).
OMe
OMe
O
O
O
(a)
(b)
OR
NHBoc
N
N
O
N
I
O
F
F
F
NHBoc
NHBoc
23: R = H (34%)
24: R = Ns (35%)
21:
22: (59%)
20
(c)
O
O
NHR
HN
NHR
HN
O
O
(d)
N
O
O
N
O
F
F
27: R = Boc
25: R = Boc
26: R = H (21% over two steps)
(e)
(e)
28: R = H (24% over two steps)
Scheme 4. Reagents and conditions: (a) Et3N, CH2Cl2, 0 °C, 48 h (59%); (b) LiBH4, THF, 0 °C, 4 h then rt, 20 h (34%); (c) 3-nitrobenzenesulphonyl chloride, Et3N, CH2Cl2, 0 °C 4 h
then rt, 20 h (35%); (d) 5-hydroxy-2H-1,4-benzoxazin-3 (4H)-one or 8-hydroxy-quinolin-2 (H)-one, Cs2CO3, DMF, rt, 36 h (e) TFA, CH2Cl2, rt, 1 h (21% over two steps).
O
O
O
O
R3
R2
O
HN
R3
R2
HN
HN
R1
HO
O
(a)
(b)
R2
R1
R1
29: R1 = OMe, R2 = H
30: R1 = R2 = H
32: R1 = OMe, R2 = H, R3 = NHMe
33: R1 = H, R2 = H, R3 = pyrrolidin
36: R1 = OMe, R2 = H, R3 = NHMe
37: R1 = H, R2 = H, R3 = pyrrolidin
38: R1 = OPMB, R2 = Cl, R3 = NH(cPr)
39: R1 = OMe, R2 = H, R3 = NH(cPr)
31: R1 = OPMB, R2 = Cl
34: R1 = OPMB, R2 = Cl, R3 = NH(cPr)
35: R1 = OMe, R2 = H, R3 = NH(cPr)
O
(c)
OH
HN
R3
O
N
O
X
R2
R1
40-50
Scheme 5. Reagents and conditions: (a) MeNH2, EtOH, rt, 20 h or pyrrolidine, 80 °C, 4 h or cyclopropylamine, rt, 6 h or cyclopropylamine 50 °C, 3 h; (b) 2(S)-oxiran-2-yl-
methyl-3-nitrobenzenesulphonate, Cs2CO3, DMF, rt, 20–48 h; (c) spirocyclic amine 2 or 20, EtOH, 80 °C (17–98%), Table 1.
with 8-hydroxyquinolin-2 (1H)-one in the presence of Cs2CO3 in
DMF gave 27 which was treated with trifluoroacetic acid to give
28 in 24% isolated yield over two steps.
The urea derivatives were synthesised as described in Scheme 5
and Table 1. Compounds 29, 30 and 31 were treated separately
with methylamine, pyrrolidine or cyclopropylamine, neat or in

N. Hossain et al. / Bioorg. Med. Chem. Lett. 24 (2014) 108–112
111
Table 1
For Scheme 5
compounds revealed that, whilst 4 was a potent CCR1 antagonist,
5 and 6 lost potency to some extent compared to 1a–d. These com-
pounds were found to have good permeability but were metaboli-
cally unstable and potent inhibitors of hERG.12 These shortcomings
could potentially be addressed by the incorporation of a polar het-
erocycle fused to the phenyl ring, thus compound 9 was designed
and synthesised. However, biological evaluation revealed that 9
was a much less potent CCR1 antagonist. An alternative approach
was to replace the carbon linker hydroxyl group of fused bicyclic
compounds 4 and 6 with a primary amino group as in 1c. Thus
fused bicyclic compounds 26 and 28 were designed and synthes-
ised. In vitro biological evaluation revealed that both compounds
lost CCR1 binding affinity relative to the corresponding alcohol
compounds, with 28 having an IC50 of only 500 nM. Finally, to fur-
ther investigate the effect of a five membered heterocycle fused
with a phenyl ring, 17–19 were designed and synthesised. These
compounds were found to be inactive in the CCR1 binding affinity
assay.
Entry
X
R1
R2
R3
Yield (%)
H3C
N
H
40
41
F
F
OCH3
OH
H
H
64
45
H3C
N
H
N
42
Cl
H
H
52
H3C
H3C
N
H
43
44
Cl
Cl
OCH3
OH
H
H
66
17
N
H
N
45
46
47
48
49
50
Cl
Cl
Cl
Cl
F
OPMB
OH
Cl
Cl
H
H
H
H
H
N
H
65
78
53
79
37
N
H
OCH3
OH
N
H
In view of the limited CCR1 binding affinity13 of the fused bicy-
clic compounds attention was turned to the urea derivatives
which, as potentially more metabolically stable alternatives to
the amide compounds, would be expected to exhibit reduced toxic
aniline metabolite formation. From this series, compounds 40 and
41 exhibited high CCR1 binding affinity with 41 being fourfold
more potent than 40. Moreover, 41 was stable in human micro-
somes with moderate hERG activity. However, this compound
was less stable in human hepatocytes and had low permeability
as measured in the Caco-2 assay. Replacement of the fluoro substi-
tuent on the spirocyclic moiety of 40 and 41 with chloro gave 43
and 44, respectively. These compounds were also potent CCR1
antagonists and additionally 44 was metabolically stable both in
human microsomes and hepatocytes, with good permeability.
However, compound 44 was unfortunately also active against the
hERG channel. The introduction of a para chloro group on the
substituted phenol and replacement of the urea methyl substituent
with a cyclopropyl group gave 46 which, whilst being potent CCR1
antagonist, was regretably less stable in human hepatocytes. How-
ever, 46 was found to be the most potent antagonist of rodent
CCR1 in this series of compounds with an IC50 value of 16 nM in
the rat binding assay. The removal of the para chloro on the substi-
tuted phenol of 46 led to 48 which was not only a very potent
antagonist of human and rat CCR1, but also metabolically stable
with modest permeability. However, the hERG channel activity
was regrettably also high for this compound. A pyrrolidine urea
derivative 42 was designed to investigate the effect of additional
substitution on the urea moiety, the meta hydroxyl group on the
substituted phenol being removed in this compound. The CCR1
N
H
OCH3
OH
N
H
F
ethanol to give 32–35 in high yields. These intermediates were
converted to the corresponding epoxides 36–39 by treatment with
(2S)-oxiran-2-yl-methyl-3-nitrobenzene sulphonate in the pres-
ence of Cs2CO3 in DMF in high yields. The epoxides 36–39 were
opened with spirocyclic amines in ethanol to afford 40, 43, 45,
47 and 49 in good yields. The methyl group was removed from
40, 43, 47 and 49 by treatment with BBr3 in dichloromethane, to
afford the hydroxyl compounds 41, 44, 46 and 50, respectively in
moderate yields. The para-methoxybenzyl group in 45 was re-
moved by brief treatment with TFA, to afford 46 in 65% isolated
yield over two steps.
Due to the poor reactivity of 29–31 with ammonia, the primary
urea derivatives 57–59 were obtained from the alternative route
outlined in Scheme 6. The N-acetyl compounds 51–53, prepared
following procedures10 previously described for 1a–d, were hydro-
lysed, by treatment with hydrochloric acid to give aniline deriva-
tives 54–56 in quantitative yield. Compounds 54, 55 and 56 were
treated separately with KOCN in the presence of NaOAc, acetic acid
and water to give 57, 58 and 59, respectively in high yields.
Initial attempts to mask the N-acetyl moiety of 1a–d in a cyclic
form, to minimize the toxic aniline metabolites formation, led to
the fused bicyclic molecules 4, 5 and 6. In vitro data for these
O
OH
NH2
R1
OH
HN
O
O
(a)
N
O
N
O
Cl
Cl
51: R1 = OH
52: R1 = H
53: R1 = F
54: R1 = OH (100%)
55: R1 = H (100%)
56: R1 = F (100%)
R1
O
OH
HN
NH2
O
(b)
N
O
Cl
57: R1 = OH (87%)
58: R1 = H (79%)
59: R1 = F (69%)
R1
Scheme 6. Reagents and conditions: (a) Aqueous HCl (1 M), 100 °C, 24 h; (b) NaOAc, KOCN, AcOH, H2O (69–100%).

112
N. Hossain et al. / Bioorg. Med. Chem. Lett. 24 (2014) 108–112
Table 2
In vitro biological data13
Entry
hCCR1 MIP-1
a, IC50
(l
M)
rCCR1 MIP-1
a, IC50(l
M)
hmics
ll/min/mg
hheps
l
l/min/106cells
Caco-2 cm/s  106
hERG IC50 (lM)
4
5
6
9
0.0071 0.001
0.032 n.a
0.037 0.031
0.56 0.275
1.26
n.a
n.a
>6
n.a
n.a
n.a
n.a
n.a
33.7
65
12.5
n.a
15
n.a
7.8
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
10.7
n.a
n.a
5.4
20.7
n.a
6.4
n.a
5.3
<3.8
<3
11
16
0.6
0.5
0.6
n.a
n.a
n.a
n.a
1.1
1.2
n.a
13.1
n.a
n.a
4.6
1
0.7
2.4
n.a
3.8
>8.9
0.4
0.4
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
0
3.5
n.a
9
n.a
n.a
2.67
n.a
0.93
0.5
14
17
18
19
26
28
40
41
42
43
44
46
47
48
49
50
57
58
59
0.16
0
1.3 1.26
1.3 0.424
n.a
0.079
0
<10
<10
87.9
<10
>200
71.2
<10
<11.4
114
<10
133
15.1
<10
21.5
17
0.56 0.275
0.013
0
1.78
n.a
n.a
0.0032 0.001
0.0014 0.0002
0.0045 0.0007
0.0013 0.0009
0.0018 0.0003
0.0018 0.0003
0.00089 0.0005
0.0022 0.0003
0.447
1.76
0.016
0.05
0.05
0.09
0.101
1.58
2.11
1.62
0.00063
0
0.0032 0.0014
0.0067 0.0008
0.0056 0.0009
5.7
16
binding affinity of 42 was retained but it was unfortunately not
Acknowledgements
stable in human liver microsomes.
hheps: human hepatocytes (
rheps: rat hepatocytes (
l/min/106cells)
l/min/mg)
l
l/min/106cells)
We thank our colleagues at the Departments of Biosciences and
DMPK for generating data, Annika U. Petersson for proof reading
this manuscript. We also thank Dr. Peter Sjö, Director of Medicinal
Chemistry, for some suggestions in writting this manuscript.
l
hmics: human microsomes (
l
Caco-2: human colonic adenocarcinoma (cm/s  106)
hERG: human Ether-a-go-go-Related-Gene
MIP-1a: macrophage inflammatory protein-1-alfa
References and notes
Compounds 49 and 50, the corresponding fluoro-substituted
spirocycle derivatives of 47 and 48, respectively, were found to
have good CCR1 binding affinity with 50 being the most potent
CCR1 antagonist of this series and with good rodent cross-over.
However, this compound had only modest metabolic stability,
was less permeable than 48 and active against the hERG channel.
Finally, the investigation was extended to unsubstituted urea
derivatives 57–59 which were also found to be very potent CCR1
antagonists. Additionally, compounds 58 and 59 exhibited good
permeability and good stability in human hepatocytes but unfortu-
nately hERG channel activity was high for these compounds. On
the contrary, 57, whilst being stable both in human hepatocytes
and microsomes, exhibited low activity against the hERG channel.
However, the permeability of 57 as measured in the Caco2 assay
was low (see Table 2).
In conclusion, a series of fused bicyclic and urea derivatives
were designed, synthesised and evaluated in vitro as very potent
CCR1 antagonists. Fused bicyclic derivative 4 was a potent CCR1
antagonist with good permeability and modest metabolic stabil-
ity. In general, the urea derivatives were very potent and of par-
ticular interest were compounds 44, 48 and 50 which combined
potent hCCR1 antagonism with good metabolic stability and per-
meability. Often, species cross-over is a challenge in the develop-
ment of chemokine receptor modulators making it difficult to
evaluate the pharmacodynamic properties of the compounds in
animal models and thereby predict the potential clinical rele-
vance of the mechanism of chemokine receptor antagonism.
However, this investigation showed that many of the compounds
in this series did exhibit good species cross-over, with 46 being a
particularly potent rat CCR1 antagonist. Thus, some of these com-
pounds, especially 46 and 48 may in fact be expected to show
efficacy in rodent models of inflammatory diseases. Unfortu-
nately, these compounds also have a tendency to inhibit the hERG
channel.
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